Chromosome aberrations induced by protons up to 31 MeV in cultured human cells

Summary Chromosome aberrations were induced in cultured human cells by proton beams of 31, 12, and 8 MeV. The frequencies of isocromatid breaks and dicentrics have been analysed as a function of proton energy and dose. Both effects are largely dependent on proton energy; isochromatid breaks increase linearly with the dose, whereas dicentrics show a definite parabolic behaviour. The experimental data were fitted to the analytic formY = KD ⁿ andY = D +D ² and the best fitted values of the parameters are reported and discussed. The values of RBE for the isochromatid breaks are in the ratio 1.7: 1.3: 1 for 8, 12, and 31 MeV respectively.In the case of the dicentrics the RBE values are dose-dependent function of the typeCD ^–n . The three distributions of dicentrics among the cells do not fit a Poisson distribution.Supported by a grant of CNR n^o 790067996 of the Finalized Project Tumour Growth Control201D; Consiglio Nazionale delle Ricerche, Italy     

Multinucleate cells and micronucleus formation in cultured human cells exposed to 12 MeV protons and gamma-rays

Cultured human cells of the EUE line were exposed to different doses of 12 MeV protons, plated and allowed to grow for 8 days; colonies were then scored for the presences of multinucleate cells and micronuclei. The frequency of both effects is an increasing function of the dose; the evaluated exponents of the dose-response equation (e = bDn) are n = 1.0 %/- 0.1 for multinucleate cells and n = 1.6 +/- 0.1 for micronuclei. By comparison with the results obtained with gamma irradiations, r.b.e. values were obtained for both effects. The correlation between the logarithm of the surviving fraction and the yield of the studied effects has been proved to be statiscally significant.     

Eremothecium ashbyii mutants resistant to 8-azaguanine. II. Mutants with different degrees of resistance to 8-azaguanine]

Guanine, unlike adenine and hypoxanthine, can not eliminate the inhibitory effect of adenine analogues on the growth and flavinogenesis of Eremothecium ashbyii. Guanine does not restore riboflavin synthesis inhibited with 5-10(-3) M 8-azaguanine. Low adenine concentrations (10(-4)-3-10(-4) M), which do not influence the inhibitory effect of 5.-10(-3) M 8-azaguanine, restore the riboflavin synthesis in combination with guanine. On the basis of the data obtained as well as the data of biochemical analysis it is concluded that the riboflavin producer studied lacks guanosinemonophosphate reductase. The mutants resistant to various concentrations of 8-azaguanine have been obtained. In all mutants resistant to 8-azaguanine the efficiency of the incorporation of 14C-guanine and 14C-adenine into mycelium is decreased as compared with the susceptible strain. The mutant Azg-R 10 resistant to high (3-10(-3) M) concentrations of 8-azaguanine, 8-azaadenine and 2,6-diaminopurine secretes inosine-like compounds when grown in a synthetic medium. The stepwise increase of the mutant resistance to 8-azaguanine from 10(-4) M TO 3-10(-3) M did not result in further enhancement of riboflavin synthesis.     

Mutagenesis in cultured human diploid cells. IV. Induction of 8-azaguanine resistant mutations by phloxine, a mutagenic red dye.

Disodium 9-(3',4',5',6'-tetrachloro-o-carboxyphenyl)-6-hydroxy-2,4,5,7-tetrabromo-3-isoxanthone (phloxine), a red dye used as a food additive, was tested for its activity to induce 8-azaguanine (8AG) resistant mutations in cultured human embryonic cells. Phloxine had a severe cytotoxic effect on the cells at concentrations of 1 to 10 mug/ml. At concentrations of more than 30 mug/ml of phloxine no further decrease in cell survival was found. This cytotoxic effect of phloxine was not dependent on the duration of treatment. After treatment with phloxine for 2 h division of cells in normal medium was inhibited for 120 h. When cells were treated with phloxine at various concentrations for 2 h, cultured in normal medium for 48 h, and then selected with 30 mug/ml of 8AG, an increase in the induced mutation frequency was found. This increase in mutation frequency was dependent on the concentration of phloxine used as a mutagen and treatment with 100 mug/ml of phloxine increased the frequency to six times that in untreated cultures.     

Anomalies in the selection of mutants of Schizosaccharomyces pombe resistant to 8-azaguanine

Summary Anomalies in selection render 8-azaguanine unsuitable as a selective agent for screening forward mutations in continuous cultures of Sch. pombe. This system may, however, provide the basis for an enrichment method for the simultaneous isolation of mutants at two loci.     

X-ray-induced mutants resistant to 8-azaguanine. II. Cell cycle dose response.

Synchronous Chinese hamster ovary cells were irradiated in G1 or S phase. Colony survival in Alpha MEM medium with dialyzed serum was determined with or without 15 mug/ml 8-azaguanine (AG). An expression period of over three generations (multiplicity of 20) was utilized, with expression times ranging from 58 to 114 h. Both G1 and S phase were practically identical in sensitivity to X-ray-induced mutations, with mutant frequency/viable cell/rad ranging from 1 X 10(-7) (75-100 rad) to 8 X 10(-7) (1000 rad). The spontaneous mutation rate, shown by Luria-Delbruck fluctuation analysis, was 5 X 10(-7) per generation. Thirty-three mutants, isolated at random and grown for over 30 generations in the absence of AG, were analyzed for plating efficiency (PE) in different concentrations of AG or in hypoxanthine-aminopterin-thymidine (HAT) medium. Of these, 64% were resistant (PE greater than 0.1) to 7.5 mug/ml AG, 85% to 5.0 mug/ml, and 91% to 3.5 mug/ml. Only 42% showed possible hypoxanthine-phosphoribosyltransferase (hprtase) deficiency as evidenced by HAT sensitivity (PE less than 0.1). Wild type controls exhibited PE's in 3.5 mug/ml AG of less than 0.001 and in HAT of greater than 0.5. Of ten mutants studied, all demonstrated survival response to radiation similar to wild type cells (D0 of approx. 120 rad). For radiation protection standards, the radiation dose required to induce mutations at a rate equal to that occurring spontaneously is called the doubling dose. The doubling dose observed for acute irradiation was about 3 rad and was estimated to be 10-60 rad for chronic irradiation, similar to that often reported for in vivo studies.     

X-ray-induced mutants resistant to 8-azaguanine. I. Effects of cell density and expression time.

Asynchronous Chinese hamster ovary cells were irradiated and colony survival in Alpha MEM medium with dialyzed serum was determined with or without 15 mug/ml 8-Azaguanine (AG). Data indicated that a reproducible assay for the system was dependent upon controlling cell density at least two days prior to induction as well as throughout the expression period. Generally, spontaneous and radiation-induced mutant frequencies decreased when cell densities exceeded a critical density of 3-6 X 10(4) cells/cm2. Infrequently, the critical density was exceeded by a factor of two with no observed decrease, possibly correlated with a longer cell doubling time. Drug depletion artifacts can occur because of drug degradation, or because wild-type cells utilize the drug or produce conditions which reduce uptake of the drug. Thus, as the effective drug concentration is lowered, the observed mutant frequency increases because a spectrum of mutants resistant to only low concentrations can now survive. In fact, refeeding with AG at intervals during the incubation period lowered spontaneous and radiation-induced frequencies approx. 5-fold. Therefore, to standardize conditions, cells were trypsinized at the end of the expression time and replated at a constant cell number for mutant selection by AG. Over two generations of growth during the expression period were required for optimal manifestation of induced mutants, and when densities were kept below 4 X 10(4) cells/cm2 at all times, observed mutant frequencies did not change significantly over a period between 80 and 140 h post-induction (over 4 generations for irradiated cells and over 6 generations for controls). Previous reports of observed mutant frequencies decreasing beyond three generations may be due to cell interaction prior to mutant selection.     

The relative effectiveness of 6-thioguanine and 8-azaguanine in selecting resistant mutants from two V79 Chinese hamster cells in vitro

The frequency of surviving colonies in two V79 cell lines exposed to either 6-thioguanine or 8-azaguanine was dependent on initial plating density. Different degrees of metabolic-co-operation were found to occur in the two cell lines and the loss of both spontaneous and added mutants occurred at a lower cell density when 6TG was used for selection than when 8 AZ was used in both cell lines. Both analogues were degraded on incubation in medium plus serum in the absence of added cells. Variation in serum batch had little effect on the rate of degradation or on the frequency of colonies recovered after treatment of V79 cell lines with 8AZ. The reasons for preferring 8AZ to 6TG as a selective agent are discussed.     

Induction of mutants resistant to antibiotics inBrevibacterium flavum

The optimum conditions for the induction of mutants resistant to antibiotics in Brevibacterium flavum ATCC 14067 were determined. UV irradiation at the energy fluence of 6.5 kJ/m2 and N-methyl-N'-nitro-N-nitrosoguanidine (1 mg/mL) at pH 6.0 were used for the induction of mutants. Mutant strains resistant to rifampicin, oleandomycin, streptomycin and erythromycin were prepared.